1) Last year, we reported that phosphorylation of the serines in the non-helical tailpiece of Acanthamoeba myosin II modifies its filament structure. The bipolar, antiparallel minifilaments of phosphorylated myosin have longer bare zones than non-phosphorylated myosin between the heads at the opposite ends of the filaments, and the heads at each end are more tightly clustered than in non-phosphorylated myosin. These results have significant implications for the regulation of the polymerization and filament structure of the three mammalian non-muscle myosin IIs as their non-helical tailpiece and adjacent coiled-coil region have multiple serines and threonines that are phosphorylated both in vitro and in vivo. Therefore, we have initiated studies on the effect of phosphorylation on the critical concentrations (the monomer concentration in equilibrium with filaments) and filament structures of the three mammalian NMIIs. This year we obtained the control data for non-phosphorylated recombinant myosins. The heavy chains of NMIIA, NMIIB and NMIIC with N-terminal FLAG tags were separately expressed in Sf9 cells together with the regulatory and essential light chains (RLC and ELC), and the proteins were purified to electrophoretic homogeneity by affinity chromatography. Their critical concentrations were determined by light scattering and ultracentrifugation after polymerization overnight at 0 degrees in 10 mM MOPS, pH 7.0, 150 mM NaCl, 0.1 mM EGTA, 2 mM MgCl2, with and without addition of 1 mM ATP, and before and after phosphorylation of the regulatory light chain. The critical concentrations under all conditions were significantly different for the three recombinant myosins than the values reported previously for myosins purified from thymus and intestinal brush border. A paper reporting our results is in preparation. 2) There is a variety of evidence for the functional association of NMIIs with biological membranes, but there is evidence both for and against the direct association of NMIIs with the membrane lipids. There are minimal, but positive, data for association of NMIIs with liposomes, but there are no data using pure NMIIA, NMIIB and NMIIC, and no data on the molecular basis of the binding of these myosins to phospholipids. We found that pure, recombinant full length NMIIA, NMIIB and NMIIC bind to 100% phosphatidylserine liposomes but not to 100% phosphatidylcholine liposomes. Binding to phosphatidylcholine liposomes containing various concentrations of either phosphatidylserine (PS) or phosphatidylinositol-4,5-diphosphate (PIP2) was proportional to the net negative charge of the liposomes with no specificity for PIP2 vs. PS. Contrary to previous proposals, from studies of the association of myosin rods with liposomes, we found that deletion of the non-helical tailpiece did not affect binding of the recombinant NMIIs to liposomes. A paper reporting these and related results is in preparation.